Fructose-1,6-bisphosphate prevents pregnancy loss by inducing decidual COX-2+ macrophage differentiation.

Decidualization is an intricate biological process in which extensive remodeling of the endometrium occurs to support the development of an implanting blastocyst. However, the immunometabolic mechanisms underlying this process are still largely unknown. We found that the decidualization process is accompanied by the accumulation of fructose-1,6-bisphosphate (FBP). The combination of FBP with pyruvate kinase M stimulated IL-27 secretion by endometrial stromal cells in an ERK/c-FOS-dependent manner. IL-27 induced decidual COX-2+ M2-like macrophage differentiation, which promotes decidualization, trophoblast invasion, and maternal-fetal tolerance. Transfer of Ptgs2+/COX-2+ macrophages prevented fetal loss in Il27ra-deleted pregnant mice. FBP levels were low in plasma and decidual tissues of patients with unexplained recurrent spontaneous abortion. In therapeutic studies, FBP supplementation significantly improved embryo loss by up-regulation of IL-27-induced COX-2+ macrophage differentiation in a mouse model of spontaneous abortion. These findings collectively provide a scientific basis for a potential therapeutic strategy to prevent pregnancy loss.

Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners.

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion. Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety. Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-ß) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss. Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

IL-2 and IL-27 synergistically promote growth and invasion of endometriotic stromal cells by maintaining the balance of IFN-γ and IL-10 in endometriosis.

Immune cells and cytokines have important roles in the pathogenesis of endometriosis. However, the production and role of cytokines of T helper type 1 (Th1) and Th2 cells in the progress of endometriosis have remained to be fully elucidated. The present study reported that the interferon (IFN)-γ levels and the percentage of IFN-γ+CD4+ cells were significantly increased in the peritoneal fluid (PF) at the early stage and maintained at a higher level at the advanced stage of endometriosis; furthermore, interleukin (IL)-10 and IL-10+CD4+ cells were elevated in the advanced stage of endometriosis. In addition, IL-2 levels in the PF at the advanced stage of endometriosis were elevated and negatively associated with IFN-γ expression. In a co-culture system of ectopic endometrial stromal cells (ESCs) and macrophages, elevated IL-2 was observed, and treatment with cytokines IL-2 and transforming growth factor-ß led to upregulation of the ratio of IL-2+ macrophages. IL-27-overexpressing ESCs and macrophages were able to induce a higher ratio of IL-10+CD4+ T cells. Blocking of IL-2 with anti-IL-2 neutralizing antibody led to upregulation of the ratio of IFN-γ+CD4+ T cells in the co-culture system in vitro. Recombinant human IL-10 and IFN-γ promoted the viability, invasiveness and transcription levels of matrix metalloproteinase (MMP)2, MMP9, and prostaglandin-endoperoxide synthase 2 of ESCs, particularly combined treatment with IL-10 and IFN-γ. These results suggest that IL-2 and IL-27 synergistically promote the growth and invasion of ESCs by modulating the balance of IFN-γ and IL-10 and contribute to the progress of endometriosis.

Cyclooxygenase-2 in Endometriosis.

Endometriosis (EMS) is the most common gynecological disease in women of reproductive age, and it is associated with chronic pelvic pain, dyspareunia and infertility. As a consequence of genetic, immune and environmental factors, endometriotic lesions have high cyclooxygenase (COX)-2 and COX-2-derived prostaglandin E2 (PGE2) biosynthesis compared with the normal endometrium. The transcription of the PTGS2 gene for COX-2 is associated with multiple intracellular signals, which converge to cause the activation of mitogen-activated protein kinases (MAPKs). COX-2 expression can be regulated by several factors, such as estrogen, hypoxia, proinflammatory cytokines, environmental pollutants, metabolites and metabolic enzymes, and platelets. High concentrations of COX-2 lead to high cell proliferation, a low level of apoptosis, high invasion, angiogenesis, EMS-related pain and infertility. COX-2-derived PGE2 performs a crucial function in EMS development by binding to EP2 and EP4 receptors. These basic findings have contributed to COX-2-targeted treatment in EMS, including COX-2 inhibitors, hormone drugs and glycyrrhizin. In this review, we summarize the most recent basic research in detail and provide a short summary of COX-2-targeted treatment.

Anti-inflammatory cytokines in endometriosis.

Although the pathogenesis of endometriosis is not fully understood, it is often considered to be an inflammatory disease. An increasing number of studies suggest that differential expression of anti-inflammatory cytokines (e.g., interleukin-4 and -10, and transforming growth factor-ß1) occurs in women with endometriosis, including in serum, peritoneal fluid and ectopic lesions. These anti-inflammatory cytokines also have indispensable roles in the progression of endometriosis, including by promoting survival, growth, invasion, differentiation, angiogenesis, and immune escape of the endometriotic lesions. In this review, we provide an overview of the expression, origin, function and regulation of anti-inflammatory cytokines in endometriosis, with brief discussion and perspectives on their future clinical implications in the diagnosis and therapy of the disease.

Estrogen restricts the apoptosis of endometrial stromal cells by promoting TSLP secretion.

Endometriosis (EMS) is a female hormone­ dependent disease with controversial reports of its etiology and pathogenesis. Apoptosis is particularly important in the human endometrium due to the dynamic cycles of proliferation and shedding. Estrogen possessed antiapoptotic effects on endometrial stromal cells (ESCs), which appears to be exacerbated in women with EMS; however, the underlying mechanism of the antiapoptotic effects of estrogen on ESC remains unknown. The present study aimed to determine whether estrogen regulates the apoptosis of ESCs via thymic stromal lymphopoietin (TSLP) and the associated mechanism. An ELISA was conducted to detect TSLP content in the ESC culture medium treated with estrogen. Subsequently, the early apoptotic rate and expression of B­cell lymphoma (Bcl­2) of ESCs were analyzed by flow cytometry in the presence of recombinant human TSLP, anti­human TSLP neutralizing antibody or estrogen. In the present study, it was reported that ESCs exhibited basal TSLP secretion in the absence of estrogen as reported in previous studies, and that estrogen promoted TSLP secretion of ESCs in a dose­dependent manner. The results demonstrated that estrogen suppressed the apoptosis of ESCs associated with the promotion of Bcl­2 expression, which may be partly reversed by inhibiting TSLP. Therefore, the findings of the present study revealed a novel mechanism of estrogen­dependent apoptotic suppression of ESCs associated with TSLP secretion and Bcl­2 regulation. Endogenous and estrogen­induced endometrial TSLP may promote the initiation and development of EMS via the inhibition of apoptosis.

Suppression of autophagy and HCK signaling promotes PTGS2high FCGR3- NK cell differentiation triggered by ectopic endometrial stromal cells.

Impaired NK cell cytotoxic activity contributes to the local dysfunctional immune environment in endometriosis (EMS), which is an estrogen-dependent gynecological disease that affects the function of ectopic endometrial tissue clearance. The reason for the impaired cytotoxic activity of NK cells in an ectopic lesion microenvironment (ELM) is largely unknown. In this study, we show that the macroautophagy/autophagy level of endometrial stromal cells (ESCs) from EMS decreased under negative regulation of estrogen. The ratio of peritoneal FCGR3- NK to FCGR3+ NK cells increases as EMS progresses. Moreover, the autophagy suppression results in the downregulation of HCK (hematopoietic cellular kinase) by inactivating STAT3 (signal transducer and activator of transcription 3), as well as the increased secretion of the downstream molecules CXCL8/IL8 and IL23A by ESCs, and this increase induced the upregulation of FCGR3- NK cells and decline of cytotoxic activity in ELM. This process is mediated through the depression of microRNA MIR1185-1-3p, which is associated with the activation of the target gene PTGS2 in NK cells. FCGR3- NK with a phenotype of PTGS2/COX2high IFNGlow PRF1low GZMBlow induced by hck knockout (hck-/-) or 3-methyladenine (3-MA, an autophagy inhibitor)-stimulated ESCs accelerates ESC's growth both in vitro and in vivo. These results suggest that the estrogen-autophagy-STAT3-HCK axis participates in the differentiation of PTGS2high IFNGlow PRF1low GZMBlow FCGR3- NK cells in ELM and contributes to the development of EMS. This result provides a scientific basis for potential therapeutic strategies to treat diseases related to impaired NK cell cytotoxic activity. ABBREVIATIONS: anti-FCGR3: anti-FCGR3 with neutralizing antibody; Ctrl-ESC: untreated ESCs; CXCL8: C-X-C motif chemokine ligand 8; ectoESC: ESCs from ectopic lesion; ELM: ectopic lesion microenvironment; EMS: endometriosis; ESCs: endometrial stromal cells; eutoESC:eutopic ESCs; HCK: hematopoietic cellular kinase; HCK(OE): overexpression of HCK; IFNG: interferon gamma; IL23A (OE): overexpression of IL23A; KLRK1: Killer cell lectin like receptor K1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3 -MA: 3-methyladenine; 3-MA-ESC: 3-MA-treated ESCs; MIR1185-1-3p+: overexpression of HsMIR1185-1-3p; NK: natural killer; normESCs: normal ESCs; Rap-ESC:rapamycin-treated ESCs; PCNA: proliferating cell nuclear antigen; PF: peritoneal fluid; SFKs: SRC family of cytoplasmic tyrosine kinases; si-HCK: silencing of HCK; siIL23A: silencing of IL23A; USCs: uterus stromal cells.

Rapamycin Synergizes with Cisplatin in Antiendometrial Cancer Activation by Improving IL-27-Stimulated Cytotoxicity of NK Cells.

Natural killer (NK) cell function is critical for controlling initial tumor growth and determining chemosensitivity of the tumor. A synergistic relationship between rapamycin and cisplatin in uterine endometrial cancer (UEC) in vitro has been reported, but the mechanism and the combined therapeutic strategy for endometrial cancer (EC) are still unknown. We found a positive correlation between the level of IL-27 and the differentiated stage of UEC. The increase of IL-27 in uterine endometrial cancer cell (UECC) lines (Ishikawa, RL95-2 and KLE) led to a high cytotoxic activity of NK cells to UECC in the co-culture system. Exposure with rapamycin enhanced the cytotoxicity of NK cells by upregulating the expression of IL-27 in UECC and IL-27 receptors (IL-27Rs: WSX-1 and gp130) on NK cells and further restricted the growth of UEC in Ishikawa-xenografted nude mice. In addition, treatment with rapamycin resulted in an increased autophagy level of UECC, and IL-27 enhanced this ability of rapamycin. Cisplatin-mediated NK cells' cytotoxic activity and anti-UEC activation were independent of IL-27; however, the combination of rapamycin and cisplatin led to a higher cytotoxic activity of NK cells, smaller UEC volume and longer survival rate in vivo. These results suggest that rapamycin and cisplatin synergistically activate the cytotoxicity of NK cells and inhibit the progression of UEC in both an IL-27-dependent and -independent manner. This provides a scientific basis for potential rapamycin-cisplatin combined therapeutic strategies targeted to UEC, especially for the patients with low differentiated stage or abnormally low level of IL-27.

Human thymic stromal lymphopoietin promotes the proliferation and invasion of cervical cancer cells by downregulating microRNA-132 expression.

Thymic stromal lymphopoietin (TSLP), produced by cervical cancer (CC) cells, promotes angiogenesis, and the recruitment and functional regulation of eosinophils. It has been reported that microRNA (miR)-132 is aberrantly decreased in CC tissues. However, the function and mechanism of TSLP on the biological behaviors of CC cells is largely unknown. The aim of the present study was to investigate the effect of TSLP on the expression of miR-132 and the proliferation and invasion in vitro of CC cell lines, namely, HeLa and SiHa cells. The transcrpitional level of miR-132 was analyzed using reverse transcription-quantitative polymerase chaon reaction. The proliferation, invasion, and the expression of proliferation and invasion-related molecules in HeLa and SiHa cells in vitro were evaluated using bromodeoxyuridine cell proliferation, Matrigel invasion assays, flow cytometry and ELISA, respectively. Here, it was revealed that recombinant human TSLP (rhTSLP) downregulated the expression levels of miR-132 in HeLa and SiHa cells, and by contrast, the neutralizing antibodies for TSLP or TSLP receptor (TSLPR) upregulated miR-132 expression levels in HeLa and SiHa cells. The overexpression of miR-132 resulted in a lowered proliferation and invasiveness, decreased levels of proliferation-associated molecules marker of proliferation Ki-67 and proliferating cell nuclear antigen, and the decreased production of matrix metalloproteinase (MMP)2 and MMP9 in HeLa and SiHa cells. Compared with the control group, there was a higher level of proliferation and invasion in HeLa and SiHa cells following stimulation with rhTSLP. However, these effects induced by rhTSLP were significantly impaired in HeLa and SiHa cells with miR-132 overexpression. The results of the present study indicated that TSLP produced by CC cells downregulated miR-132 expression, and stimulated the proliferation and invasion of CC cells, thereby further promoting the development of CC.

Autophagy in endometriosis.

Endometriosis (EMS) is a common gynecologic disease that causes chronic pelvic pain, dysmenorrhea, and infertility in women. The doctrine of menstruation back flow planting and defects in the immune system are well known and widely accepted. In recent years, increasing studies have been focused on the role of autophagy in EMS, and have shown that autophagy plays a vital role in EMS. Autophagy, which is known as the non-apoptotic form of programmed cell death induced by a large number of intracellular/extracellular stimuli, is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles in eukaryotic cells. Autophagy commonly refers to macroautophagy, which is characterized by autophagosomes (double-membrane vesicles). In normal endometrial tissues, autophagy is induced in glandular epithelial and stromal cells throughout the menstrual cycle. However, aberrant autophagy occurs in the eutopic endometrium and ectopic endometriotic foci, which contributes to the pathogenesis of EMS by promoting the hyperplasia of endometriotic tissues and stromal cells, restricting apoptosis, and inducing abnormal immune responses. Consistent with changes in autophagy levels between normal endometria, eutopic and ectopic endometria from patients with EMS, the altered expression of autophagy-related genes (ATGs) is also observed. Currently, many factors are involved in the aberrant autophagy of endometriotic tissues, including female hormones, certain drugs, hypoxia, and oxidative stress. Therefore, studies focusing on autophagy may uncover a new potential treatment for EMS. The aim of this review is to discuss the role of aberrant autophagy in EMS and to explore the potential value of autophagy as a target for EMS therapy.

The crosstalk between endometrial stromal cells and macrophages impairs cytotoxicity of NK cells in endometriosis by secreting IL-10 and TGF-ß.

The dysfunction of NK cells in women with endometriosis (EMS) contributes to the immune escape of menstrual endometrial fragments refluxed into the peritoneal cavity. The reciprocal communications between endometrial stromal cells (ESCs) and lymphocytes facilitate the development of EMS. However, the mechanism of these communications on cytotoxicity of natural killer (NK) cells in endometriotic milieus is still largely unknown. To imitate the local immune microenvironment, the co-culture systems of ESCs from patients with EMS and monocyte-derived macrophages or of ESCs, macrophages and NK cells were constructed. The cytokine levels in the co-culture unit were evaluated by ELISA. The expression of functional molecules in NK cells was detected by flow cytometry (FCM). The NK cell behaviors in vitro were analyzed by cell counting kit-8 and cytotoxic activation assays. After incubation with ESCs and macrophages, the expression of CD16, NKG2D, perforin and IFN-γ, viability and cytotoxicity of NK cells were significantly downregulated. The secretion of interleukin (IL)-1ß, IL-10 and transforming growth factor (TGF)-ß in the co-culture system of ESCs and macrophages was increased. Exposure with anti-IL-10 receptor ß neutralizing antibody (αhIL-10Rß) or αTGF-ß could partly reverse these effects of ESCs and macrophages on NK cells in vitro These results suggest that the interaction between macrophages and ESCs downregulates cytotoxicity of NK cells possibly by stimulating the secretion of IL-10 and TGF-ß, and may further trigger the immune escape of ectopic fragments and promote the occurrence and the development of EMS.

RANKL-mediated harmonious dialogue between fetus and mother guarantees smooth gestation by inducing decidual M2 macrophage polarization.

Decidual macrophages (dMϕ) contribute to maternal-fetal tolerance. However, the mechanism of dMϕ differentiation during pregnancy is still largely unknown. Here, we report that receptor activator for nuclear factor-κ B ligand (RANKL), secreted by human embryonic trophoblasts and maternal decidual stromal cells (DSCs), polarizes dMϕ toward a M2 phenotype. This polarization is mediated through activation of Akt/signal transducer and activator of transcription 6 (STAT6) signaling, which is associated with the upregulation of histone H3 lysine-27 demethylase Jmjd3 and IRF4 in dMϕ. Such differentiated dMϕ can induce a Th2 bias that promotes maternal-fetal tolerance. Impaired expression of RANKL leads to dysfunction of dMϕ in vivo and increased rates of fetal loss in mice. Transfer of RANK+Mϕ reverses mouse fetal loss induced by Mϕ depletion. Compared with normal pregnancy, there are abnormally low levels of RANKL/RANK in villi and decidua from miscarriage patients. These results suggest that RANKL is a pivotal regulator of maternal-fetal tolerance by licensing dMϕ to ensure a successful pregnancy outcome. This observation provides a scientific basis on which a potential therapeutic strategy can be targeted to prevent pregnancy loss.

Immunosuppressive macrophages induced by IDO1 promote the growth of endometrial stromal cells in endometriosis.

It was previously demonstrated that anomalous expression of indoleamine 2,3-dioxygenase-1 (IDO1) in endometrial stromal cells (ESCs) stimulated an inflammatory response that subsequently initiated the activation of immunosuppressive macrophages in endometriosis. The aim of the present study was to clarify the effect of IDO1­induced macrophages on the growth of ESCs in endometriosis. Normal ESCs, ectopic ESCs and normal ESCs treated with plasmid pEGFP­N1­IDO1 or SD11­IDO1 short hairpin RNA were co­cultured with peripheral blood­derived monocyte (PBMC)­driven macrophages directly for 48 h. Compared with normal ESCs, the PBMC­driven macrophages that were co­cultured with ectopic ESCs displayed a lower phagocytic ability. pEGFP­N1­IDO1 transfection of normal ESCs also decreased the phagocytic ability of co­cultured macrophages. Additionally, pEGFP­N1­IDO1­transfected ESC­induced macrophages significantly increased the viability and proliferation of ESCs, while ESC apoptosis was decreased, compared with control ESCs. In conclusion, IDO1 educated-macrophages may facilitate the survival of retrograde endometrial tissues, and be involved in the pathogenesis of endometriosis.

IL-27 triggers IL-10 production in Th17 cells via a c-Maf/RORγt/Blimp-1 signal to promote the progression of endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease. The anti-inflammatory cytokine IL-10 is also increased in endometriosis. IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. However, the mechanism of inducing IL-10-producing Th17 cells is still largely unknown. The present study investigated the differentiation mechanism and role of IL-10-producing Th17 cells in endometriosis. Here, we report that IL-10+Th17 cells are significantly increased in the peritoneal fluid of women with endometriosis, along with an elevation of IL-27, IL-6 and TGF-ß. Compared with peripheral CD4+ T cells, endometrial CD4+ T cells highly expressed IL-27 receptors, especially the ectopic endometrium. Under external (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and local (estrogen, IL-6 and TGF-ß) environmental regulation, IL-27 from macrophages and endometrial stromal cells (ESCs) induces IL-10 production in Th17 cells in vitro and in vivo. This process may be mediated through the interaction between c-musculoaponeurotic fibrosarconna (c-Maf) and retinoic acid-related orphan receptor gamma t (RORγt), and associated with the upregulation of downstream B lymphocyte-induced maturation protein-1 (Blimp-1). IL-10+Th17 cells, in turn, stimulate the proliferation and implantation of ectopic lesions and accelerate the progression of endometriosis. These results suggest that IL-27 is a pivotal regulator in endometriotic immune tolerance by triggering Th17 cells to produce IL-10 and promoting the rapid growth and implantation of ectopic lesions. This finding provides a scientific basis for potential therapeutic strategies aimed at preventing the development of endometriosis, especially for patients with high levels of IL-10+Th17 cells.

TSLP promotes angiogenesis of human umbilical vein endothelial cells by strengthening the crosstalk between cervical cancer cells and eosinophils.

Our previous study demonstrated that thymic stromal lymphopoietin (TSLP) secreted by cervical cancer cells promotes angiogenesis and recruitment, and regulates the function of eosinophils (EOS). However, the function of TSLP in the crosstalk between EOS and vascular endothelial cells in cancer lesions remains unknown. The aim of the present study was to investigate the effect of EOS caused by TSLP in in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). The results of the present study revealed that recombinant human TSLP protein (rhTSLP) increased the secretion of vascular endothelial growth factor (VEGF), but not fibroblast growth factors, in HL-60-eosinophils (HL-60E). Compared with cervical cancer cells (HeLa or CasKi cells) or HL-60E alone, there were increased levels of interleukin (IL)-8 and VEGF in the co-culture system between cervical cancer cells, and HL-60E cells. This effect was strengthened by rhTSLP, but inhibited by inhibiting the TSLP signal with anti-human TSLP or TSLP receptor neutralizing antibodies. The results of the tube formation assays revealed that treatment with the supernatant from cervical cancer cells and/or HL-60E resulted in an increase in angiogenesis in HUVECs, which could be decreased by TSLP or TSLPR inhibitors. The results of the present study suggested that TSLP derived of cervical cancer cells may indirectly stimulate angiogenesis of HUVECs, by upregulating IL-8 and VEGF production, in a co-culture model between cervical cancer cells and EOS, therefore promoting the development of cervical cancer.

Macrophages promote the growth and invasion of endometrial stromal cells by downregulating IL-24 in endometriosis.

Macrophages play an important role in the origin and development of endometriosis. Estrogen promoted the growth of decidual stromal cells (DSCs) by downregulating the level of interleukin (IL)-24. The aim of this study was to clarify the role and mechanism of IL-24 and its receptors in the regulation of biological functions of endometrial stromal cells (ESCs) during endometriosis. The level of IL-24 and its receptors in endometrium was measured by immunohistochemistry. In vitro analysis was used to measure the level of IL-24 and receptors and the biological behaviors of ESCs. Here, we found that the expression of IL-24 and its receptors (IL-20R1 and IL-20R2) in control endometrium was significantly higher than that in eutopic and ectopic endometrium of women with endometriosis. Recombinant human IL-24 (rhIL-24) significantly inhibited the viability of ESCs in a dosage-dependent manner. Conversely, blocking IL-24 with anti-IL-24 neutralizing antibody promoted ESCs viability. In addition, rhIL-24 could downregulate the invasiveness of ESCs in vitro After co-culture, macrophages markedly reduced the expression of IL-24 and IL-20R1 in ESCs, but not IL-22R1. Moreover, macrophages significantly restricted the inhibitory effect of IL-24 on the viability, invasion, the proliferation relative gene Ki-67, proliferating cell nuclear antigen (PCNA) and cyclooxygenase2 (COX-2), and the stimulatory effect on the tumor metastasis suppressor gene CD82 in ESCs. These results indicate that the abnormally low level of IL-24 in ESCs possibly induced by macrophages may lead to the enhancement of ESCs' proliferation and invasiveness and contribute to the development of endometriosis.

IL15 promotes growth and invasion of endometrial stromal cells and inhibits killing activity of NK cells in endometriosis.

Endometriosis (EMS) is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissues. It has been reported that human endometrial stromal cells (ESCs) express interleukin (IL)15. The aim of our study was to elucidate whether or not IL15 regulates the cross talk between ESCs and natural killer (NK) cells in the endometriotic milieu and, if so, how this regulation occurs. The ESC behaviors in vitro were verified by Cell Counting Kit-8 (CCK-8), Annexin/PI, and Matrigel invasion assays, respectively. To imitate the local immune microenvironment, the co-culture system between ESCs and NK cells was constructed. The effect of IL15 on NK cells in the co-culture unit was investigated by flow cytometry (FCM). In this study, we found that ectopic endometrium from patients with EMS highly expressed IL15. Rapamycin, an autophagy inducer, decreased the level of IL15 receptors (i.e. IL15Rα and IL2Rß). IL15 inhibits apoptosis and promotes the invasiveness, viability, and proliferation of ESCs. Meanwhile, a co-culture with ESCs led to a decrease in CD16 on NK cells. In the co-culture system, IL15 treatment downregulated the levels of Granzyme B and IFN-γ in CD16(+)NK cells, NKG2D in CD56(dim)CD16(-)NK cells, and NKP44 in CD56(bright)CD16(-)NK cells. On the one hand, these results indicated that IL15 derived from ESCs directly stimulates the growth and invasion of ESCs. On the other hand, IL15 may help the immune escape of ESCs by suppressing the cytotoxic activity of NK cells in the ectopic milieu, thereby facilitating the progression of EMS.

Blocking IL-22, a potential treatment strategy for adenomyosis by inhibiting crosstalk between vascular endothelial and endometrial stromal cells.

Our previous work has demonstrated that interleukin-22 (IL-22) enhances the invasiveness of endometrial stromal cells (ESCs) of adenomyosis in an autocrine manner. In the present study, we further investigated whether IL-22 mediated crosstalk between vascular endothelial cells (VECs) and ESCs in vitro. Here we found that VECs in ectopic lesion from women with adenomyosis highly expressed IL-22 receptors IL-22R1 and IL-10R2. Both recombinant human IL-22 (rhIL-22) and IL-22 from ESCs increased IL-22R1 and IL-10R2 expression on human umbilical vein endothelial cells (HUVECs). Treatment with rhIL-22 led to an elevation of HUVECs viability, but did not influence HUVECs apoptosis. In contrast, anti-human IL-22 neutralizing antibody (α-IL-22) inhibited HUVECs viability induced by supernatants of ESCs. Stimulation with rhIL-22 or ESCs up-regulated CD105 expression on HUVECs and promoted angiogenesis, and α-IL-22 could reverse these effect induced by ESC. Compared to non-treated HUVECs, HUVECs educated by rh-IL-22 or ESCs could further up-regulate Ki-67 and proliferating cell nuclear antigen (PCNA) expression, and down-regulate Fas ligand (FasL) expression in ESCs. However, these effects induced by ESC-educated HUVECs were inhibited by α-IL-22. These results suggest that IL-22 derived from ESC promotes IL-22 receptors expression and enhances the viability, activation and angiogenesis of HUVEC. In turn, the educated HUVEC may further stimulate proliferation and restricts apoptosis of ESC. The integral effect may contribute to the progress of adenomyosis. Blocking IL-22 can disturb crosstalk between ESC and VEC mediated by IL-22, suggesting that blocking IL-22 may be a potential treatment strategy for adenomyosis.

Chemokine CCL17 induced by hypoxia promotes the proliferation of cervical cancer cell.

Cervical cancer is often associated with hypoxia and many kinds of chemokines. But the relationship and role of hypoxia and Chemokine (C-C motif) ligand 17 (CCL17) in cervical cancer are still unknown. Here, we found that CCL17 was high expressed in cervical cancer. HeLa and SiHa cells could secrete CCL17 in a time-dependent manner. Hypoxia increased expression of CCL17 receptor (CCR4) on HeLa and SiHa cells. Treatment with recombination human CCL17 (rhCCL17) led to an elevation of cell proliferation in HeLa and SiHa cells in a dose-dependent manner. In contrast, blocking CCL17 with anti-human CCL17 neutralizing antibody (α-CCL17) played an oppose effect. However, rhCCL17 had no effect on apoptosis in cervical cancer cells. Further analysis showed that hypoxia promoted the proliferation of HeLa and SiHa cells, and these effects could be reversed by α-CCL17. Stimulation with the inhibitor for c-Jun N-terminal kinase (JNK) or signal transducers and activator of transcription 5 (STAT5) signal pathway not only directly decreased the proliferation of HeLa and SiHa cells, but also abrogated the stimulatory effect of rhCCL17 on the proliferation of HeLa and SiHa cells. These results suggest that a high level of CCL17 in cervical cancer lesions is an important regulator in the proliferation of cervical cancer cells through JNK and STAT5 signaling pathways. In this process, hypoxia magnifies this effect by up-regulating CCR4 expression and strengthening the interaction of CCL17/CCR4.

RANKL/RANK interaction promotes the growth of cervical cancer cells by strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion.

Receptor activator for nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family. The interaction between RANKL and its receptor RANK plays an important role in the development and function of diverse tissues. However, the expression and role of RANKL in cervical cancer are still unknown. In the present study, we found that RANKL and RANK were highly co-expressed in cervical cancer. HeLa and SiHa cells secreted soluble RANKL (sRANKL), expressed member RANKL (mRANKL) and RANK. Recombinant human RANKL protein had no effect on the viability of HeLa and SiHa cells. Yet, blocking RANKL with an anti-human RANKL neutralizing antibody (α-RANKL) or recombinant human osteoprotegrin (OPG) protein resulted in the downregulation of Ki-67 and B-cell lymphoma 2 (Bcl-2) expression and an increase in Fas and Fas ligand (FasL) expression, as well as a high level of viability and a low level of apoptosis in the HeLa and SiHa cells. In addition, α-RANKL led to a decrease in IL-8 secretion. Recombinant human IL-8 protein reversed the effect of α-RANKL on the expression of proliferation- and apoptosis­related molecules, and proliferation and apoptosis in the HeLa and SiHa cells. The present study suggests that a high level of mRANKL/RANK expression in cervical cancer lesions plays an important role in the rapid growth of cervical cancer cells possibly through strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion, which may be a possible target for cervical cancer therapy.